Xingnaojing Regulates The Mechanism Of HIF-1α Signaling Pathway To Reduce The Secondary Damage Of Cerebral Hemorrhage

Background:Intracerebral hemorrhage(ICH)is one of the most common acute cerebral vascular diseases,characterized by acute,rapid progress,and cerebral hernia and other serious complications can occur in a short time,with high mortality and morbidity.After ICH,blood accumulated in the parenchyma rapidly,causing the destruction of normal anatomic structures and the increase of the local pressure,resulting in secondary injury and produce a series of pathophysiological changes,including decreased blood flow perfusion,edema formation and change of permeability of blood-brain barrier around hematoma(Blood-brain barrier,BBB)and tissue injury,etc.ICH also causes anoxia and ischemia in brain tissue,resulting overproduction and accumulation of reactive oxygen species(ROS),thereby induces oxidative stress of nerve cells.Oxidative stress result in a variety of pathological changes of brain tissue,such as cell apoptosis autophagy inflammation,and blood-brain barrier damage,etc..Hypoxia of the brain tissue induces upregulation of hypoxia inducible factor(HIF).The expression of HIF induces the expression of a series of downstream gene transcription,playing an important role in ICH.The pathway of HIF and downstream gene BNIP3 is important in autophagy and apoptosis,which regulates the process of brain injury after ICH.Xingnaojing(XNJ)injection is derived from a traditional Chinese medicine Angongniuhuang pill,with natural musk borneol,gardenia and Yujin as the main component.XNJ injection effectively promote the absorption of intracranial hematoma in ICH patients,reduce inflammation and cerebral edema,and improve neurological function,but the mechanism by which it protect brain injury after ICH is unknown.This study used the collagenase-induced rat ICH model,combined with the pathological imaging and molecular biology methods to observe hemorrhage rats brain tissue ultrastructure change and relevant pathological changes,and detect the expression of key protein molecules,including HIF-1a,BBB related proteins,autophagy related proteins and apoptosis related proteins,after the cerebral ischemic hypoxia changes,so as to explore the mechanism that how XNJ injection protect the secondary injury after ICH.Objective:1 To detect neurological defects and edema formation in a rat ICH model and evaluate the protective effect of Xingnaojing injection on brain tissue after cerebral hemorrhage.2 To detect the effect of Xingnaojing injection on the expression of proteins related to BBB,autophagy and apoptosis and explore the mechanism by which Xingnaojing injection in regulation of autophagy,apoptosis and theh permeability of BBB,in order to provide theoretical and experimental basis for the early intervention of ICH with Chinese medicine under the guidance of the theory of "Disease collaterals-toxin damaging brain collaterals".Methods:1 A model of intracerebral hemorrhage was produced by injection of collagenase VII into the caudatum of rats.Healthy male SD rats of SPF grade(8 weeks)were divided into four groups by using random number table:the sham group,the model group,and the Xingnaojing group and the edaravone Group.Nerve function as evaluated by mNSS score in the rats.after hemorrhage rats for three days later,snapping the beheading of rats brain,respectively,by HE staining,Ni’s staining and transmission electron microscopy(TEM)were used to detect edema formation,blood brain barrier change and nerve cells apoptosis after three days treatment.3 Evens blue(EB)method was used to detect the BBB permeability.Immunohistochemical was used to detect the expression of ZO-1,Occludin,AQP4,MMP9 and aotuphagy-relating proteins Beclin-land BNIP3.Western blot was used to detect the expression of ZO-1,Occludin,AQP4,VEGF,autophagy-relating proteins including Beclin-1,BNIP3,LC3,and apoptosis-relating proteins including Bcl-2 and Bax.Results:1 mNSS score showed that the neurological function score of rats in the model group of cerebral hemorrhage was significantly higher than that in the sham group,and the neurological function was obviously impaired.Compared with the model group,the neural function scores of Xingnaojing group and edaravone group were significantly decreased,and the neural function defects were significantly improved.2 HE stanning,Nissl stanning and EB stanning showed that compared with the control group,the rats in the model group had an obvious increase of the permeability of blood-brain barrier(BBB)as well as increased bleeding and edema area.Nerve cell morphological was changed obviously and brain injury was severe.TEM results showed that the structure of astrocytes and vascular endothelial cells was disrupted markedly in model group.Compare to the model group,rats in the Xingnaojing and edaravone groups showed a low permeability of the BBB,decrease bleeding and edema area;The structure of neuronal astrocytes and vascular endothelial cells was intact and nerve function was improved in the Xingnaojing and edaravone groups.3 Immunohistochemical results showed that the expressions of HIF,AQP4,MMP9,VEGF,Beclin-1 and BNIP3 were significantly higher than those in the sham group,while the expressions of ZO-1 and Occludin were significantly lower than those in the sham group.Compared with the model group,the expressions of HIF,AQP4,MMP9,VEGF,Beclin-1 and BNIP3 in brain tissues of rats after cerebral hemorrhage in Xingnaojing and edaravone groups were significantly decreased,while the expressions of ZO-1 and Occludin were significantly increased.4 Western blot showed that compared with the control group,the expressions of HIF,AQP4,MMP9,VEGF,Beclin-1,BNIP3,LC3,Bax was increased significantly,and the expression of Occludin,ZO-1 and Bcl-2 was significantly reduced.Compared with model group,the expressions of HIF,AQP4,VEGF,Beclin-1,BNIP3,LC3 and Bax was decreased significantly,whilethe expression of ZO-1,Occludin,and Bcl-2 was significantly increased.Conclusion:Xingnaojing injection significantly reduce hemorrhage rats nerve function score,alleviate nerve function defect,reduce the permeability of blood brain barrier,alleviate nerve cell damage.Xingnaojing injection significantly decreases the expression of HIF and BNIP3,apoptosis and autophagy,thus plasy a critical role in neuroprotection after intracerebral hemorrhage.